Crispr editing to embed nucleic acid landing pads into genomes of live cells

ABSTRACT

The present disclosure relates to compositions, methods, modules and automated integrated instrumentation for multiplex delivery of “landing pad” edits into the genomes of a population of live cells. The landing pads then may be leveraged to insert very large DNA sequences into the genomes of the population of live cells.

RELATED CASES

This application claims priority to U.S. Ser. No. 63/078,789, filed 15Sep. 2020, entitled “CRISPR EDITING TO EMBED NUCLEIC ACID LANDING PADSINTO GENOMES OF LIVE CELLS”, which is incorporated herein in itsentirety.

FIELD OF THE INVENTION

The present disclosure relates to compositions, methods, modules andautomated integrated instrumentation for multiplex delivery of “landingpad” edits into the genome of a population of live cells.

INCORPORATION BY REFERENCE

Submitted with the present application is an electronically filedsequence listing via EFS-Web as an ASCII formatted sequence listing,entitled “INSC070US_seq_list_20210823”, created Aug. 23, 2021, and 823bytes in size. The sequence listing is part of the specification filedherewith and is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

The ability to make precise, targeted changes to the genome of livingcells has been a long-standing goal in biomedical research anddevelopment. Recently, various nucleases have been identified that allowfor manipulation of gene sequences; hence gene function. The nucleasesinclude nucleic acid-guided nucleases (i.e., CRISPR nucleases), whichenable researchers to generate permanent edits in live cells; however,currently the payload that can be inserted is approximately 100 basepairs or less.

There is thus a need in the art of nucleic acid-guided nuclease editingfor improved methods, compositions, modules and automated, integratedinstruments to facilitate insertion of payloads greater than 100 basepairs into a cellular genome by leveraging a combination of highthroughput nucleic acid-guided nuclease editing and lower-throughputrecombinase/integrase or HDR-mediated insertion. The present disclosureaddresses this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure relates to methods, compositions, modules andautomated multi-module cell processing instruments that allow one toperform nucleic acid-guided nuclease editing to embed “landing pads”into one or more—typically several to many—target loci in a populationof cells in a multiplexed manner. The landing pads then can be leveragedto insert large DNA payloads (e.g., >100 bp) into the target loci. Anadvantage of the present methods and compositions is that they allow oneto leverage CRISPR-type nucleic acid-guided nuclease genome-widetargeted editing to insert landing pads in the cellular genome, followedby insertion of large DNA sequences (e.g., >100 bp) via lower-throughputrecombinase/integrase or HDR-mediated insertions or substitutions. Thelanding pads encode an enzyme recognition sequence such as arecombinase, integrase or meganuclease recognition sequence.

Thus, there is provided a method for multiplex insertion of large DNApayloads into a population of cells and identifying cells with a desiredphenotype or genotype comprising the steps of: designing andsynthesizing a library of editing cassettes comprising landing pads,wherein the editing cassettes further comprise a gRNA comprisinghomology to a target sequence in the cells and a repair templatecomprising 5′ and 3′ homology arms flanking the landing pad; insertingthe library of editing cassettes into a vector backbone resulting in alibrary of editing vectors; transforming the population of cells withthe library of editing vectors; allowing editing to take place in thepopulation of cells to produce edited cells; transforming the editedcells with vectors carrying large DNA payloads, wherein the vectorscarrying large DNA payloads further comprise a coding sequence for arecombinase or a meganuclease under control of an inducible promoter;inducing expression of the recombinase or a meganuclease to insert thelarge DNA payloads into the landing pads; and screening for cellscomprising the desired phenotype or genotype.

In some embodiments of this method, the vectors carrying large DNApayloads comprise a coding sequence for a recombinase, the landing padscomprise a recognition sequence for the recombinase and the large DNApayloads comprise recognition sequences for the recombinase flanking thelarge DNA payload. In aspects of this embodiment, the recombinase is acyclization recombination enzyme (Cre) and the landing pad and large DNApayload comprise lox recombination sites. Alternatively, the recombinaseis flippase and the landing pad and large DNA payload comprise flippaserecognition targets (FRTs).

In yet another embodiment of this method, the vectors carrying large DNApayloads comprise a coding sequence for a meganuclease, the landing padscomprise a recognition sequence for the meganuclease, and the large DNApayloads further comprise homologous recombination sequences flankingthe DNA payloads. In some aspects, the meganuclease belongs to theLAGLIDADG family of nucleases, and in some aspects, the meganuclease isI-SceI; the meganuclease is I-CreI; or the meganuclease is I-DmoI.

In some embodiments of the method, the editing cassettes furthercomprise a barcode and/or an amplification priming site at the 3′ end ofthe editing cassette. In some embodiments, the vectors carrying thelarge DNA payloads further comprise a selectable marker and the methodfurther comprises a selection step between the transforming and allowingsteps. In some aspects, the selectable marker in the vectors carryingthe large DNA payloads is different from a selectable marker in theediting vectors.

In some aspects of the method, the vectors carrying the large DNApayloads comprise the coding sequence of the recombinase or meganucleaseunder the control of an inducible promoter. In some aspects, theinducible promoter is a pL promoter or a pBAD promoter.

In some aspects of the method, the vectors carrying large DNA payloadsfurther comprise an origin of replication and a selectable marker.

In some embodiments, the large DNA payloads are from 100 bp to 100 Kb inlength, and in some embodiments, the large DNA payloads are from 250 bpto 10 Kb in length.

In some embodiments, the screening step comprises polymerase chainreaction (PCR) analysis with appropriate primer sets; a metabolic test;measurement of transcript level; a phenotypic assay; detection of aprotein product using an antibody specific to the protein product; orDNA sequencing of the integrated large DNA payload.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1A is a simple process diagram for performing nucleic acid-guidednuclease editing in a population of cells to embed landing pads intotarget genetic loci in a population of cells, then leverage the landingpads to insert large DNA payloads (e.g., >100 bp) into the target loci.FIG. 1B is a simplified depiction of the process described in FIG. 1A.FIG. 1C is an exemplary editing cassette used to embed a landing padinto a target genome in a cell facilitating subsequent insertion of alarge payload via a sequence-specific recombinase/integrase. FIG. 1D isan exemplary editing cassette used to embed a landing pad into a targetgenome in a cell facilitating subsequent insertion of a large payloadvia a meganuclease system. FIG. 1E is an exemplary vector for insertinga large DNA sequence into an embedded landing pad via a recombinase.FIG. 1F is an exemplary vector for inserting a large DNA payload into anembedded landing pad via a meganuclease.

FIGS. 2A-2C depict three different views of an exemplary automatedmulti-module cell processing instrument for performing nucleicacid-guided nuclease editing.

FIGS. 3A-3C depict various components of exemplary embodiments of abioreactor module included in an integrated instrument useful forgrowing and transfecting cells. FIGS. 3D and 3E depict an exemplaryintegrated instrument for growing and transfecting cells.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentof the methods, devices or instruments described herein are intended tobe applicable to the additional embodiments of the methods, devices andinstruments described herein except where expressly stated or where thefeature or function is incompatible with the additional embodiments. Forexample, where a given feature or function is expressly described inconnection with one embodiment but not expressly mentioned in connectionwith an alternative embodiment, it should be understood that the featureor function may be deployed, utilized, or implemented in connection withthe alternative embodiment unless the feature or function isincompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions ofmolecular biology (including recombinant techniques), cell biology,biochemistry, and genetic engineering technology, which are within theskill of those who practice in the art. Such conventional techniques anddescriptions can be found in standard laboratory manuals such as Greenand Sambrook, Molecular Cloning: A Laboratory Manual. 4th ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014);Current Protocols in Molecular Biology, Ausubel, et al. eds., (2017);Neumann, et al., Electroporation and Electrofusion in Cell Biology,Plenum Press, New York (1989); Chang, et al., Guide to Electroporationand Electrofusion, Academic Press, California (1992); Viral Vectors(Kaplift & Loewy, eds., Academic Press (1995)); all of which are hereinincorporated in their entirety by reference for all purposes. Formammalian/stem cell culture and methods see, e.g., Basic Cell CultureProtocols, 4th ed. (Helgason & Miller, eds., Humana Press 2005); Cultureof Animal Cells, Seventh Ed. (Freshney, ed., Humana Press 2016);Microfluidic Cell Culture, Second Ed. (Borenstein, Vandon, Tao &Charest, eds., Elsevier Press 2018); Human Cell Culture (Hughes, ed.,Humana Press 2011); 3D Cell Culture (Koledova, ed., Humana Press 2017);Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, eds., John Wiley & Sons 1998); Essential Stem Cell Methods,(Lanza & Klimanskaya, eds., Academic Press 2011); Essentials of StemCell Biology, 3d ed., (Lanza & Atala, eds., Academic Press 2013); andHandbook of Stem Cells, (Atala & Lanza, eds., Academic Press 2012), allof which are herein incorporated in their entirety by reference for allpurposes. CRISPR editing techniques can be found in, e.g., GenomeEditing and Engineering from TALENs and CRISPRs to Molecular Surgery,Appasani and Church (2018); and CRISPR: Methods and Protocols, Lindgrenand Charpentier (2015); both of which are herein incorporated in theirentirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TAGCTG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents—translated in an appropriate host cell.

The terms “editing cassette”, “CREATE cassette”, “CREATE editingcassette”, “CREATE fusion editing cassette” or “CFE editing cassette”refers to a nucleic acid molecule comprising a coding sequence fortranscription of a guide nucleic acid or gRNA covalently linked to acoding sequence for transcription of a repair template.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the repair template witha certain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

A “landing pad” is a sequence of nucleotides inserted into a genome orepisome of a cell via CRISPR editing comprising a recognition sequence.

The term “meganuclease” refers to an endodeoxyribonuclease characterizedby a large recognition site (double-stranded DNA sequences of 12 to 40base pairs) and as a result the recognition site generally occurs onlyonce, if at all, in any given genome.

“Nucleic acid-guided editing components” refers to one, some, or all ofa nucleic acid-guided nuclease or nickase fusion enzyme, a guide nucleicacid and a repair template.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

A “PAM mutation” refers to one or more edits to a target sequence thatremoves, mutates, or otherwise renders inactive a PAM or spacer regionin the target sequence.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA. Promoters may be constitutive or inducible.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably. Proteins may or may not be made up entirely of aminoacids.

“Recognition sequences” are particular sequences of nucleotides that aprotein, DNA, or RNA molecule, or combinations thereof (such as, but notlimited to, a restriction endonuclease, a modification methylase or arecombinase) recognizes and binds. For example, a recognition sequencefor Cre recombinase is a 34 base pair sequence containing two 13 basepair inverted repeats (serving as the recombinase binding sites)flanking an 8 base pair core and designated loxP (see, e.g., Sauer,Current Opinion in Biotechnology, 5:521-527 (1994)). Other examples ofrecognition sequences include, but are not limited to, attB and attP,attR and attL and others that are recognized by the recombinase enzymebacteriophage Lambda Integrase. The recombination site designated attBis an approximately 33 base pair sequence containing two 9 base paircore-type Int binding sites and a 7 base pair overlap region; attP is anapproximately 240 base pair sequence containing core-type Int bindingsites and arm-type Int binding sites as well as sites for auxiliaryproteins IHF, FIS, and Xis (see, e.g., Landy, Current Opinion inBiotechnology, 3:699-7071 (1993)).

A “recombinase” is an enzyme that catalyzes the exchange of DNA segmentsat specific recombination sites. An “integrase” refers to a recombinasethat is usually derived from viruses or transposons, as well as perhapsancient viruses. “Recombination proteins” include excisive proteins,integrative proteins, enzymes, co-factors and associated proteins thatare involved in recombination reactions using one or more recombinationsites (again see, e.g., Landy, Current Opinion in Biotechnology,3:699-707 (1993)). The recombination proteins used in the methods hereincan be delivered to a cell via an expression cassette on an appropriatevector, such as a plasmid or viral vector. In other embodiments,recombination proteins can be delivered to a cell in protein form in thesame reaction mixture used to deliver the desired nucleic acid(s). Inyet other embodiments, the recombinase could also be encoded in the celland expressed upon demand using a tightly controlled inducible promoter.

As used herein the terms “repair template” or “donor nucleic acid” or“donor DNA” or “homology arm” refer to 1) nucleic acid that is designedto introduce a DNA sequence modification (insertion, deletion,substitution) into a locus by homologous recombination using nucleicacid-guided nucleases, or 2) a nucleic acid that serves as a template(including a desired edit) to be incorporated into target DNA by reversetranscriptase in a CREATE fusion editing (CFE) system. Forhomology-directed repair, the repair template must have sufficienthomology to the regions flanking the “cut site” or the site to be editedin the genomic target sequence. For template-directed repair, the repairtemplate has homology to the genomic target sequence except at theposition of the desired edit although synonymous edits may be present inthe homologous (e.g., non-edit) regions. The length of the repairtemplate(s) will depend on, e.g., the type and size of the modificationbeing made. In many instances and preferably, the repair template willhave two regions of sequence homology (e.g., two homology arms)complementary to the genomic target locus flanking the locus of thedesired edit in the genomic target locus. Typically, an “edit region” or“edit locus” or “DNA sequence modification” region—the nucleic acidmodification that one desires to be introduced into a genome targetlocus in a cell (e.g., the desired edit)—will be located between tworegions of homology. The DNA sequence modification may change one ormore bases of the target genomic DNA sequence at one specific site ormultiple specific sites. A change may include changing 1, 2, 3, 4, 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 ormore base pairs of the target sequence. A deletion or insertion may be adeletion or insertion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 75,100, 150, 200, 300, 400, or 500 or more base pairs of the targetsequence.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampicillin/carbenicillin,kanamycin, chloramphenicol, nourseothricin N-acetyl transferase,erythromycin, tetracycline, gentamicin, bleomycin, streptomycin,puromycin, hygromycin, blasticidin, and G418 may be employed. In otherembodiments, selectable markers include, but are not limited to humannerve growth factor receptor (detected with a MAb, such as described inU.S. Pat. No. 6,365,373); truncated human growth factor receptor(detected with MAb); mutant human dihydrofolate reductase (DHFR;fluorescent MTX substrate available); secreted alkaline phosphatase(SEAP; fluorescent substrate available); human thymidylate synthase (TS;confers resistance to anti-cancer agent fluorodeoxyuridine); humanglutathione S-transferase alpha (GSTA1; conjugates glutathione to thestem cell selective alkylator busulfan; chemoprotective selectablemarker in CD34+ cells); CD24 cell surface antigen in hematopoietic stemcells; human CAD gene to confer resistance toN-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1(MDR-1; P-glycoprotein surface protein selectable by increased drugresistance or enriched by FACS); human CD25 (IL-2a; detectable byMab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable bycarmustine); rhamnose; and Cytidine deaminase (CD; selectable by Ara-C).“Selective medium” as used herein refers to cell growth medium to whichhas been added a chemical compound or biological moiety that selects foror against selectable markers.

The terms “target genomic DNA sequence”, “target sequence”, or “genomictarget locus” and the like refer to any locus in vitro or in vivo, or ina nucleic acid (e.g., genome or episome) of a cell or population ofcells, in which a change of at least one nucleotide is desired using anucleic acid-guided nuclease editing system. The target sequence can bea genomic locus or extrachromosomal locus.

The terms “transformation”, “transfection” and “transduction” are usedinterchangeably herein to refer to the process of introducing exogenousDNA into cells.

The term “variant” may refer to a polypeptide or polynucleotide thatdiffers from a reference polypeptide or polynucleotide but retainsessential properties. A typical variant of a polypeptide differs inamino acid sequence from another reference polypeptide. Generally,differences are limited so that the sequences of the referencepolypeptide and the variant are closely similar overall and, in manyregions, identical. A variant and reference polypeptide may differ inamino acid sequence by one or more modifications (e.g., substitutions,additions, and/or deletions). A variant of a polypeptide may be aconservatively modified variant. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code (e.g., anon-natural amino acid). A variant of a polypeptide may be naturallyoccurring, such as an allelic variant, or it may be a variant that isnot known to occur naturally.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, BACs, YACs, PACs, synthetic chromosomes, andthe like. In some embodiments, a coding sequence for a nucleicacid-guided nuclease is provided in a vector, referred to as an “enginevector.” In some embodiments, the editing cassette may be provided in avector, referred to as an “editing vector.” In some embodiments, thecoding sequence for the nucleic acid-guided nuclease and the editingcassette are provided in the same vector.

Nuclease-Directed Genome Editing Generally

The compositions, methods, modules and instruments described herein areemployed to allow one to perform nucleic acid nuclease-directed genomeediting (i.e., CRISPR editing) to introduce desired edits to apopulation of live cells. Specifically, the compositions, methods,modules and integrated instruments presented herein facilitate editingnucleotide sequences in a population of cells in a multiplexed andtargeted manner, including insertions of large DNA sequences (e.g., >65bp, >75 bp, or up to 100 bp—i.e., the insertion of “landing pads”), suchthat even larger insertions of nucleic acids can be made using arecombinase, an integrase or a meganuclease. An advantage of the presentmethods and compositions is that they allow one to leverage CRISPR-typenucleic acid-guided nuclease genome-wide targeted editing to insertlanding pads in a cellular genome, followed by insertion of large DNAsequences (e.g., >100 bp) via lower-throughput recombinase/integrase orHDR-mediated insertions or substitutions into the inserted landing pads.The landing pads encode an enzyme recognition sequence such as arecombinase, integrase or meganuclease recognition sequence.

In CRISPR editing generally, a nucleic acid-guided nuclease complexedwith an appropriate synthetic guide nucleic acid in a cell can cut thegenome of the cell at a desired location. The guide nucleic acid helpsthe nucleic acid-guided nuclease recognize and cut the DNA at a specifictarget sequence. By manipulating the nucleotide sequence of the guidenucleic acid, the nucleic acid-guided nuclease may be programmed totarget any DNA sequence for cleavage as long as an appropriateprotospacer adjacent motif (PAM) is nearby. In certain aspects, thenucleic acid-guided nuclease editing system may use two separate guidenucleic acid molecules that combine to function as a guide nucleic acid,e.g., a CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA).In other aspects and preferably, the guide nucleic acid is a singleguide nucleic acid construct that includes both 1) a guide sequencecapable of hybridizing to a genomic target locus, and 2) a scaffoldsequence capable of interacting or complexing with a nucleic acid-guidednuclease.

In general, a guide nucleic acid (e.g., gRNA) complexes with acompatible nucleic acid-guided nuclease and can then hybridize with atarget sequence, thereby directing the nuclease to the target sequence.A guide nucleic acid can be DNA or RNA; alternatively, a guide nucleicacid may comprise both DNA and RNA. In some embodiments, a guide nucleicacid may comprise modified or non-naturally occurring nucleotides. Incases where the guide nucleic acid comprises RNA, the gRNA may beencoded by a DNA sequence on a polynucleotide molecule such as aplasmid, linear construct, or the coding sequence may and preferablydoes reside within an editing cassette. Methods and compositions fordesigning and synthesizing editing cassettes and libraries of editingcassettes are described in U.S. Pat. Nos. 10,240,167; 10,266,849;9,982,278; 10,351,877; 10,364,442; 10,435,715; 10,465,207; 10,669,559;10,711,284; 10,731,180; and 11,078,498; all of which are incorporated byreference herein.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. The degree of complementarity between a guidesequence and the corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences. In some embodiments, a guide sequence is about or more thanabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.In some embodiments, a guide sequence is less than about 75, 50, 45, 40,35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20nucleotides in length.

In general, to generate an edit in the target sequence, thegRNA/nuclease complex binds to a target sequence as determined by theguide RNA, and the nuclease recognizes a protospacer adjacent motif(PAM) sequence adjacent to the target sequence. The target sequence canbe any polynucleotide endogenous or exogenous to the cell, or in vitro.For example, the target sequence can be a polynucleotide residing in thenucleus of the cell. A target sequence can be a sequence encoding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., a regulatorypolynucleotide, an intron, a PAM, a control sequence, or “junk” DNA).

The guide nucleic acid may be and preferably is part of an editingcassette that encodes the repair template that targets a cellular targetsequence. Alternatively, the guide nucleic acid may not be part of theediting cassette and instead may be encoded on the editing vectorbackbone. For example, a sequence coding for a guide nucleic acid can beassembled or inserted into a vector backbone first, followed byinsertion of the repair template in, e.g., an editing cassette. In othercases, the repair template in, e.g., an editing cassette can be insertedor assembled into a vector backbone first, followed by insertion of thesequence coding for the guide nucleic acid. Preferably, the sequenceencoding the guide nucleic acid and the repair template are locatedtogether in a rationally-designed editing cassette and aresimultaneously inserted or assembled via gap repair into a linearplasmid or vector backbone to create an editing vector.

The target sequence is associated with a proto-spacer mutation (PAM),which is a short nucleotide sequence recognized by the gRNA/nucleasecomplex. The precise preferred PAM sequence and length requirements fordifferent nucleic acid-guided nucleases vary; however, PAMs typicallyare 2-10 or so base-pair sequences adjacent or in proximity to thetarget sequence and, depending on the nuclease, can be 5′ or 3′ to thetarget sequence. Engineering of the PAM-interacting domain of a nucleicacid-guided nuclease may allow for alteration of PAM specificity,improve target site recognition fidelity, decrease target siterecognition fidelity, or increase the versatility of a nucleicacid-guided nuclease.

In most embodiments, genome editing of a cellular target sequence bothintroduces a desired DNA change to a cellular target sequence (an“intended” edit), e.g., the genomic DNA of a cell, and removes, mutates,or renders inactive a proto-spacer mutation (PAM) region in the cellulartarget sequence (an “immunizing edit”) thereby rendering the target siteimmune to further nuclease binding. Rendering the PAM at the cellulartarget sequence inactive precludes additional editing of the cell genomeat that cellular target sequence, e.g., upon subsequent exposure to anucleic acid-guided nuclease complexed with a synthetic guide nucleicacid in later rounds of editing. Thus, cells having the desired cellulartarget sequence edit and an altered PAM can be selected for by using anucleic acid-guided nuclease complexed with a synthetic guide nucleicacid complementary to the cellular target sequence. Cells that did notundergo the first editing event will be cut rendering a double-strandedDNA break, and thus will not continue to be viable. The cells containingthe desired cellular target sequence edit and PAM alteration will not becut, as these edited cells no longer contain the necessary PAM site andwill continue to grow and propagate.

As for the nuclease component of the nucleic acid-guided nucleaseediting system, a polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcell types, such as bacterial, yeast, and mammalian cells. The choice ofthe nucleic acid-guided nuclease to be employed depends on many factors,such as what type of edit is to be made in the target sequence andwhether an appropriate PAM is located close to the desired targetsequence. CRISPR nucleases of use in the methods described hereininclude but are not limited to Cas 9, Cas 12/CpfI, MAD2, or MAD7, MAD2007 or other MADzymes and MADzyme systems (see U.S. Pat. Nos.9,982,279; 10,337,028; 10,435,714; 10,011,849; 10,626,416; 10,604,746;10,665,114; 10,640,754; 10,876,102; 10,883,077; 10,704,033; 10,745,678;10,724,021; 10,767,169; and 10,870,761 for sequences and other detailsrelated to engineered and naturally-occuring MADzymes).

Another component of the nucleic acid-guided nuclease system is therepair template comprising homology to the cellular target sequence. Forthe present methods and compositions, the repair template typically ison the same vector and in the same editing cassette as the guide nucleicacid and is under the control of the same promoter as the editing gRNA(that is, a single promoter driving the transcription of both theediting gRNA and the repair template). The repair template is designedto serve as a template for homologous recombination with a cellulartarget sequence nicked or cleaved by the nucleic acid-guided nuclease asa part of the gRNA/nuclease complex. A repair template polynucleotidemay be of any suitable length, such as about or more than about 20, 25,50, or 75 nucleotides in length. In certain preferred aspects, therepair template can be provided as an oligonucleotide of between 20-100nucleotides, more preferably between 30-75 nucleotides. The repairtemplate comprises two regions that are complementary to a portion ofthe cellular target sequence (e.g., homology arms) flanking the mutationor difference between the repair template and the cellular targetsequence. When optimally aligned, the repair template overlaps with (iscomplementary to) the cellular target sequence by, e.g., about 20, 25,30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides. In the presentmethods and compositions, the repair template comprises at least onealteration compared to the cellular target sequence, such as a landingpad insertion compared to the cellular target sequence.

As described in relation to the gRNA, the repair template is provided aspart of a rationally-designed editing cassette, which is inserted intoan editing plasmid backbone where the editing plasmid backbone maycomprise a promoter to drive transcription of the editing gRNA and therepair template when the editing cassette is inserted into the editingplasmid backbone. Moreover, there may be more than one, e.g., two,three, four, or more editing gRNA/repair template rationally-designedediting cassettes inserted into an editing vector targeting differentregions of the genome; alternatively, a single rationally-designedediting cassette may comprise two to several editing gRNA/repairtemplate pairs targeting different regions of the genome, where eachediting gRNA is under the control of separate different promoters,separate like promoters, or where all gRNAs/repair template pairs areunder the control of a single promoter. In some embodiments the promoterdriving transcription of the editing gRNA and the repair template (ordriving more than one editing gRNA/repair template pair) is optionallyan inducible promoter.

In addition to the repair template, an editing cassette may comprise oneor more primer binding sites. The primer binding sites are used toamplify the editing cassette by using oligonucleotide primers asdescribed infra and may be biotinylated or otherwise labeled. In thecurrent embodiments, the editing cassettes are a library of editingcassettes for, e.g., inserting a single landing pad into differenttarget locations in the population of cells. Other embodiments envisionperforming successive rounds of editing where different landing pads areinserted throughout the genome of a population of cells; that is, inround 1, landing pad 1 is inserted, in round 2, landing pad 2 isinserted, and so on. In addition, the library of editing cassettes iscloned into vector backbones where, e.g., each different repair templatemay be associated with a different barcode. Also, in preferredembodiments, an editing vector or plasmid encoding components of thenucleic acid-guided nuclease system further encodes a nucleicacid-guided nuclease comprising one or more nuclear localizationsequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more NLSs, particularly as an element of the nucleasesequence. In some embodiments, the engineered nuclease comprises NLSs ator near the amino-terminus, NLSs at or near the carboxy-terminus, or acombination.

Inserting and Leveraging Genomic Landing Pads

The present disclosure relates to methods, compositions, modules andautomated multi-module cell processing instruments that allow one toperform nucleic acid-guided nuclease editing to embed “landing pads”into target loci in a population of cells in a multiplexed manner.Typically, a single landing pad type (e.g., a recognition sequence for asingle enzyme) will be inserted into different loci in different cellsresulting in a population of cells each with a landing pad inserted intoa target region where the target regions are different in differentcells. The landing pads then can be leveraged to insert large DNApayloads (e.g., >100 bp) into the target loci. An advantage of thepresent methods and compositions is that it allows one to leverageCRISPR-type nucleic acid-guided nuclease genome-wide targeted editing toinsert landing pads in the cellular genome, followed by insertion oflarge DNA payloads (e.g., >100 bp) via lower-throughputrecombinase/integrase or HDR-mediated insertions or substitutions. Thelanding pads encode an enzyme recognition sequence such as arecombinase, integrase or meganuclease recognition sequence.

FIG. 1A is a simple process diagram for performing nucleic acid-guidednuclease editing in a population of cells to embed landing pads intotarget genetic loci in a population of cells, then leverage the landingpads to insert large DNA payloads (e.g., >100 bp) into the target loci.In a first step 102 of method 100, a library of editing cassettescomprising paired gRNAs and repair templates is designed andsynthesized. The editing cassettes each comprise a gRNA comprising botha guide and a spacer designed to target a specific locus in the cellulargenome; a 5′ homology arm; a recombinase recognition site ormeganuclease recognition site; and a 3′ homology arm (the recognitionsequence forms the landing pad and the 5′ homology arm, recognitionsequence and 3′ homology arm collectively form the landing pad repairtemplate); and other desired sequences such as a barcode, primeramplification sites and the like. The various components of exemplaryediting cassettes are described in more detail infra in relation toFIGS. 1C and 1D.

Once designed and synthesized 102, the library of editing cassettes isamplified (e.g., using primer amplification sites in the editingcassettes), purified and inserted 104 into a vector backbone—which insome embodiments may already comprise a coding sequence for the nucleicacid-guided nuclease—to produce a library of editing vectors.Alternatively, the coding sequence for the nuclease may be located onanother vector that may be transformed into the cells before, at thesame time as or after the editing vectors are transformed into thecells. In yet other alternatives, the coding sequence for the nucleasemay be integrated into the cellular genome or the nuclease may bedelivered to the cell as a protein. The vectors chosen for the methodsherein will vary depending on the type of cells being edited andanalyzed, where the vectors include, e.g., plasmids, BACs, YACs, viralvectors and synthetic chromosomes.

The cells of interest useful in the methods herein are any cells,including bacterial, yeast and animal (including mammalian) cells.Before being transformed by the editing vectors, the cells are oftengrown in culture for several passages. Cell culture is the process bywhich cells are grown under controlled conditions, almost always outsidethe cell's natural environment. For bacterial and yeast cells, the cellsare typically grown in a defined medium in bulk culture. For mammaliancells, culture conditions typically vary somewhat for each cell type butgenerally include a medium and additives that supply essential nutrientssuch as amino acids, carbohydrates, vitamins, minerals, growth factors,hormones, and gases such as, e.g., O₂ and CO₂. In addition to providingnutrients, the medium typically regulates the physio-chemicalenvironment via a pH buffer and most cells are grown at 37° C. Manymammalian cells require or prefer a surface or artificial substrate onwhich to grow (e.g., adherent cells), whereas other cells such ashematopoietic cells and some adherent cells can be grown in or adaptedto grow in suspension. Adherent cells often are grown in 2D monolayercultures in petri dishes or flasks, but some adherent cells can grow insuspension cultures to higher density than would be possible in 2Dcultures. “Passages” generally refers to transferring a small number ofcells to a fresh substrate with fresh medium, or, in the case ofsuspension cultures, transferring a small volume of the culture to alarger volume of medium.

The cells of choice are provided and are transformed with the library ofediting vectors 106. The library of editing vectors comprises vectorbackbones each “carrying” at least one editing cassette. For singleedits where one landing pad is inserted per cell, the edit (e.g.,landing pad) is the same for every cassette in the library, but thelanding pad is targeted to different locations around the genome. Thelibrary of editing cassettes may have tens, hundreds, thousands, tens ofthousands or more different editing cassettes (in this case, tens,hundreds, thousands, tens of thousands or more different guides), wherethe landing pad sequences are the same but the gRNA and homology armsare different for insertion into different genomic target loci.

As used herein, transformation is intended to generically include avariety of art-recognized techniques for introducing an exogenousnucleic acid sequence (e.g., an engine and/or editing vector) into atarget cell, and the term “transformation” as used herein includes alltransformation and transfection techniques. Such methods include, butare not limited to, electroporation, lipofection, optoporation,injection, microprecipitation, microinjection liposomes, particlebombardment, sonoporation laser-induced poration bead transfection,calcium phosphate or calcium chloride co-precipitation, orDEAE-dextran-mediated transfection. Cells can also be prepared forvector uptake using, e.g., a sucrose, sorbitol or glycerol wash.Additionally, hybrid techniques that exploit the capabilities ofmechanical and chemical transfection methods can be used. e.g.,magnetofection, a transfection methodology that combines chemicaltransfection with mechanical methods. In another example, cationiclipids may be deployed in combination with gene guns or electroporators.Suitable materials and methods for transforming or transfecting targetcells can be found, e.g., in Green and Sambrook, Molecular Cloning: ALaboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2014).

Once transformed 106, the cells are allowed to recover and selectionoptionally is performed to select for cells transformed with the editingvector, which most often comprises a selectable marker. At a next step108, editing is allowed to take place. If one or both components of theediting machinery (e.g., editing cassette and nuclease) is under thecontrol of an inducible promoter, conditions are provided to induceediting. If none of the components of the editing machinery are underthe control of an inducible promoter, editing proceeds immediately aftertransformation. During the editing process, many cells may die due todouble-strand breaks in the genome that are a consequence of the editingprocess. Of the cells that do survive editing and continue to grow, thesurviving cells will comprise a landing pad that allows for insertionsor substitutions of large payloads.

After editing takes place and after recovery and growth for 1-4 hours,or typically 8, 10 or 14 hours in rich medium and optional antibioticselection at 15-37° C. (depending on cell type), the cells are grown andprepared for another round of transformation, this time with a plasmidor vector carrying 1) a coding sequence for the appropriaterecombinase/integrase or meganuclease targeting the landing padrecognition sequence; and 2) either a large payload sequence flanked byeither the recombinase or integrase recognition sequence forrecombinase/integrase-mediated insertion into the landing pad in thegenome, or a large payload sequence flanked by homology arm sequencesfor HDR-mediated insertion into the genome via the meganuclease 110.FIGS. 1E and 1F described infra depict the payload vectors in moredetail. Typically the recombinase or meganuclease is under the controlof an inducible promoter such that the expression of the recombinase ormeganuclease is tightly controlled. At step 112, expression of therecombinase or meganuclease is induced, thereby inducing the delivery ofthe large payload to the landing pad.

FIG. 1B is a simplified diagram of the process described in FIG. 1A. Themethod begins with multiplexed CRISPR-based editing of a cell populationusing editing vectors comprising the CREATE editing cassettes,preferably in an automated manner using an instrument (depicted at left)such as described U.S. Pat. Nos. 10,253,316; 10,329,559; 10,323,242;10,421,959; 10,465,185; 10,519,437; 10,584,333; 10,584,334; 10,647,982;10,689,645; 10,738,301; 10,738,663; 10,947,532; 10,894,958; 10,954,512;and 11,034,953; and U.S. Ser. No. 17/239,540. Again, the library ofediting cassettes may have tens, hundreds, thousands, tens of thousandsor more different editing cassettes, where the landing pad sequences arethe same but the gRNA and repair templates are different for insertioninto different genomic target loci. After editing, the population ofcells comprise a genome with landing pads inserted into different lociaround the genome, depicted as a black bar on a circular genome in thecell.

Following insertion of the landing pads into various loci in the genome,the cells are then transformed with a plasmid or other vector carryingthe payload to be delivered to the landing pads (depicted as stripedbars on the vectors in the cells). As described above, transformation isintended to generically include a variety of art-recognized techniquesfor introducing an exogenous nucleic acid sequence (e.g., an engineand/or editing vector) into a target cell, and the term “transformation”as used herein includes all transformation and transfection techniques.Each plasmid or vector comprises 1) a coding sequence for theappropriate recombinase/integrase or meganuclease targeting the landingpad recognition sequence; and 2) either a large payload sequence flankedby either the recombinase or integrase recognition sequence forrecombinase/integrase-mediated insertion into the landing pad in thegenome, or a large payload sequence flanked by homology arm sequencesfor HDR-mediated insertion into the genome via the meganuclease. In anoptional (but preferred) step, the plasmid or vector also comprises acoding sequence for a selection marker and the cells are selected aftertransformation.

After transformation and optional selection, delivery of the largepayloads to the landing pads in the cells is induced by inducingexpression of the recombinase/integrase or meganuclease. The cells withthe payload delivered to the landing pads are allowed to recover andgrow and then are screened. Note that after delivery of the payload tothe landing pads, the black bar on the chromosome in the cells istransformed into a striped bar. Screening for proper integration of thepayload includes but is not limited to 1) polymerase chain reaction(PCR) analysis with appropriate primer sets used to assess whether thedelivery vector was correctly integrated at the target site; 2)assessment of activity of the nucleic acid of interest, including butnot limited to a metabolic test, measurement of transcript level, aphenotypic assay, or detection of a protein product using an antibodyspecific to the protein product; and/or 3) DNA sequencing of theintegrated payload. Exemplary applications of the present compositionsand methods include genome-wide delivery of large-insert promoterlibraries; delivery of heterologous genes or pathways to a large numberof genomic locations enabling examination of location-dependentexpression effects; and delivery of fusion-protein partners to multipleloci around the genome.

FIG. 1C is an exemplary editing cassette used to embed a landing padinto a target genome in a cell allowing insertion of a large payload viaa sequence-specific recombinase/integrase. In the sequence-specificrecombinase/integrase embodiment, the editing cassette (e.g., CREATEcassette) comprises from 5′ to 3′ 1) the gRNA (guide+spacer); and 2) arepair template comprising a 5′ homology arm “wing”; a sequencerecognized by a recombinase or integrase (in this case, a loxP sequencerecognized by the Cre recombinase); a 3′ homology arm “wing”; and a “P2”priming site. Optionally, the editing cassette may also comprise abarcode positioned between the 3′ homology arm “wing” and the “P2”priming site. The sequence recognized by the recombinase or integrase isthe landing pad and the combination of the 5′ homology arm “wing”, thesequence recognized by a recombinase or integrase, and the 3′ homologyarm “wing” is the “repair template” or “landing pad repair template”which is inserted or substituted into the target sequence in thecellular genome.

Site/sequence-specific recombination differs from general homologousrecombination in that short specific DNA sequences, which are requiredfor recognition by a recombinase, are the only sites at whichrecombination occurs. Depending on the orientations of these sites on aparticular DNA strand or chromosome, the specialized recombinases thatrecognize these specific sequences can catalyze 1) DNA excision or 2)DNA inversion or rotation. Site-specific recombination can also occurbetween two DNA strands if these sites are not present on the samechromosome. A number of bacteriophage- and yeast-derived site-specificrecombination systems—each comprising a recombinase and specific cognatesites—have been shown to work in both prokaryotic and eukaryotic cellsincluding the bacteriophage P1 Cre/lox system, yeast FLP-FRT system, andthe Dre system of the tyrosine family of site-specific recombinases.Such systems and methods of use are described, e.g., in U.S. Pat. Nos.7,422,889; 7,112,715; 6,956,146; 6,774,279; 5,677,177; 5,885,836;5,654,182; and 4,959,317, each of which is incorporated herein byreference to teach methods of using such recombinases. Other systemsthat may be utilized in the compositions and methods described hereininclude those of the tyrosine family of site-specific recombinases suchas bacteriophage lambda integrase, HK2022 integrase; as well as systemsbelonging to the separate serine family of recombinases such asbacteriophage phiC31 and R4Tp901 integrases.

The methods of the invention preferably utilize combined variants of thesequence-specific recombination sites that are recognized by the samerecombinase for recombinase-mediated cassette exchange (RMCE). RMCE is aconvenient method for genetic manipulation; however, in order to achieverecombination in a desired direction, recognition site mutants are used.Examples of such sequence-specific recombination site variants includethose that contain a combination of inverted repeats or those whichcomprise recombination sites having mutant spacer sequences. Forexample, two classes of variant recombinase sites are available toengineer stable Cre-loxP integrative recombination. Both exploitsequence mutations in the Cre recognition sequence, either within the 8bp spacer region or the 13 bp inverted repeats. Spacer mutants such aslox511 (Hoess, et al., Nucleic Acids Res, 14:2287-2300 (1986)), lox5171and lox2272 (Lee and Saito, Gene, 216:55-65 (1998)), m2, m3, m7, and m11(Langer, et al., Nucleic Acids Res, 30:3067-3077 (2002)) recombinereadily with themselves but have a markedly reduced rate ofrecombination with the wild-type site. This class of mutants has beenexploited for DNA insertion by RMCE using non-interacting Cre-Loxrecombination sites and non-interacting FLP recombination sites (Baerand Bode, Curr Opin Biotechnol, 12:473-480 (2001); Albert, et al., PlantJ, 7:649-659 (1995); Seibler and Bode, Biochemistry, 36:1740-1747(1997); Schlake and Bode, Biochemistry, 33:12746-12751 (1994)). Forexample, exemplary lox variant sequences are shown in Table 1:

TABLE 1 Lox Variant Spacer sequence Lox2272 AAGTATCC Lox5171 ATGTGTACLoxM2 AGAAACCA LoxP ATGTATGC

FIG. 1D is an exemplary editing cassette used to embed a landing padinto a target genome in a cell which allows insertion of a large payloadvia a meganuclease. In the meganuclease embodiment, the editing cassette(e.g., CREATE cassette) comprises from 5′ to 3′ 1) the gRNA(guide+spacer); and 2) a repair template comprising a 5′ homology arm“wing”; a sequence recognized by a meganuclease (in this case, an I-SceIrecognition sequence); a 3′ homology arm “wing”; and a “P2” primingsite. Optionally, the editing cassette may also comprise a barcodepositioned between the 3′ homology arm “wing” and the “P2” priming site.The sequence recognized by the meganuclease is the landing pad and thecombination of the 5′ homology arm “wing”, the sequence recognized by ameganuclease, and the 3′ homology arm “wing” is the “repair template”which is inserted or substituted into the target sequence in thecellular genome.

Meganucleases are endodeoxyribonucleases characterized by a largerecognition site (double-stranded DNA sequences of 12 to 40 base pairs).As a result of the large recognition sequence, a meganucleaserecognition site generally occurs only once if at all in any givengenome. For example, the 18-base pair sequence recognized by the I-SceImeganuclease on average requires a genome twenty times the size of thehuman genome to be found once by chance (although sequences with asingle mismatch occur about three times per human-sized genome). I-SceIrecognizes an 18-base pair sequence TAGGGATAACAGGGTAAT and leaves a 4base pair 3′ hydroxyl overhang. Meganucleases are therefore consideredto be the most specific naturally-occurring restriction enzymes. Amongmeganucleases, the LAGLIDADG family of homing endonucleases has become avaluable tool for the study of genomes and genome engineering over thepast fifteen years. Meganucleases are “molecular DNA scissors” that canbe used to replace, eliminate or modify sequences in a highly targetedway. By modifying the meganuclease recognition sequence through proteinengineering, the targeted sequence can be changed. Meganucleases areused to modify all genome types, whether bacterial, plant or animal.

There are five families, or classes, of homing endonucleases; the mostwidespread and best known of which is the LAGLIDADG family. The name ofthis family corresponds to an amino acid sequence (or motif) that isfound, more or less conserved, in all the proteins of this family. Thesesmall proteins are also known for their compact and closely packedthree-dimensional structures. The best characterized endonucleases whichare most widely used in research and genome engineering include I-SceI(discovered in Saccharomyces cerevisiae), I-CreI (from the chloroplastsof the green algae Chlamydomonas reinhardtii) and I-DmoI (from thearchaebacterium Desulfurococcus mobilis). The high specificity ofmeganucleases gives them a high degree of precision and much lower celltoxicity than other naturally occurring restriction enzymes.

FIG. 1E is an exemplary vector for inserting a large DNA payload into anembedded landing pad via a recombinase (see FIG. 1C). The exemplaryvector comprises, starting at 10 o'clock and continuing clockwise, anorigin of replication for the plasmid such as a bacterial or yeastorigin of replication for propagation of the plasmid in, e.g., E. colior S. cerevisiae; a selectable marker that is preferably different thanthe selectable marker(s) used in the editing vector (and, e.g., enginevector, if used); the large insert payload flanked by recognitionsequences (in this case, loxP sites); and a coding sequence for therecombinase (here, cre) under the control of an inducible promoter.

The large payload insert is limited in size by the delivery vector butmay comprise more than 60 bp, more than 70 bp, more than 80 bp, morethan 90 bp, more than 100 bp, more than 150 bp, more than 200 bp; up to10 Kb, 15 Kb or 20 Kb for plasmids and up to 100 Kb for YAC and BACvectors; and greater for artificial chromosomes.

Expression of the recombinase is controlled preferably by an induciblepromoter to tightly control expression; examples of inducible promotersinclude the pL promoter, which is induced by an increase in temperature;the pBAD promoter, which is induced by the addition of arabinose to thegrowth medium; the tetracycline-controlled transcriptional activationsystem (Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, Calif.); Bujard andGossen, PNAS, 89(12):5547-5551 (1992)); the Lac Switch Inducible system(Wyborski et al., Environ Mol Mutagen, 28(4):447-58 (1996); DuCoeur etal., Strategies 5(3):70-72 (1992); and U.S. Pat. No. 4,833,080); theecdysone-inducible gene expression system (No et al., PNAS,93(8):3346-3351 (1996)); the cumate gene-switch system (Mullick et al.,BMC Biotechnology, 6:43 (2006)); and the tamoxifen-inducible geneexpression (Zhang et al., Nucleic Acids Research, 24:543-548 (1996)) aswell as others.

As described above, an alternative to utilizing a recombinase/integraseis utilizing a meganuclease (see FIG. 1D). FIG. 1F is an exemplaryvector for inserting a large DNA sequence into an embedded landing padvia a meganuclease. The exemplary vector comprises, starting at 10o'clock and continuing clockwise, an origin of replication for theplasmid such as a bacterial or yeast origin of replication forpropagation of the plasmid in, e.g., E. coli or S. cerevisiae; aselectable marker that is preferably different than the selectablemarkers used in the editing vector (and, e.g., engine vector, if used);the large insert payload flanked by HDR sequences compatible with theHDR sequences flanking the I-SceI recognition sequence of FIG. 1D; and acoding sequence for the recombinase (here, the I-SceI restrictionenzyme) under the control of an inducible promoter. As with the vectordepicted in FIG. 1E, the large payload insert is limited in size by thedelivery vector but may comprise more than 60 bp, more than 70 bp, morethan 80 bp, more than 90 bp, more than 100 bp, more than 150 bp, morethan 200 bp, and up to 10 Kb, 15 Kb or 20 Kb for plasmids and up to 100Kb for YAC and BAC vectors and expression of the recombinase iscontrolled preferably by an inducible promoter to tight control ofexpression.

The meganuclease cuts the landing pad and the large payload serves asthe repair template by way of its flanking arms. Yeast has a naturalrepair capability; however E. coli can be engineered to comprise, e.g.,the λ Red repair machinery to drive HDR repair.

Automated Cell Editing Instruments and Modules to Perform NucleicAcid-Guided Nuclease or Nickase Fusion Editing in Cells

One Embodiment of an Automated Cell Editing Instrument

FIG. 2A depicts an exemplary automated multi-module cell processinginstrument 200 to, e.g., perform targeted gene editing of live cells.The instrument 200, for example, may be and preferably is designed as astand-alone benchtop instrument for use within a laboratory environment.The instrument 200 may incorporate a mixture of reusable and disposablecomponents for performing the various integrated processes in conductingautomated genome cleavage and/or editing in cells without humanintervention. Illustrated is a gantry 202, providing an automatedmechanical motion system (actuator) (not shown) that supplies XYZ axismotion control to, e.g., an automated (i.e., robotic) liquid handlingsystem 258 including, e.g., an air displacement pipettor 232 whichallows for cell processing among multiple modules without humanintervention. In some automated multi-module cell processinginstruments, the air displacement pipettor 232 is moved by gantry 202and the various modules and reagent cartridges remain stationary;however, in other embodiments, the liquid handling system 258 may staystationary while the various modules and reagent cartridges are moved.Also included in the automated multi-module cell processing instrument200 are reagent cartridges 210 (see, U.S. Pat. Nos. 10,376,889;10,406,525; 10,478,822; 10,576,474; 10,639,637; 10,738,271; and10,799,868) comprising reservoirs 212 and transformation module 230(e.g., a flow-through electroporation device as described in U.S. Pat.Nos. 10,435,713; 10,443,074; and 10,851,389), as well as wash reservoirs206, cell input reservoir 251 and cell output reservoir 253. The washreservoirs 206 may be configured to accommodate large tubes, forexample, wash solutions, or solutions that are used often throughout aniterative process. Although two of the reagent cartridges 210 comprise awash reservoir 206 in FIG. 2A, the wash reservoirs instead could beincluded in a wash cartridge where the reagent and wash cartridges areseparate cartridges. In such a case, the reagent cartridge and washcartridge may be identical except for the consumables (reagents or othercomponents contained within the various inserts) inserted therein.

In some implementations, the reagent cartridges 210 are disposable kitscomprising reagents and cells for use in the automated multi-module cellprocessing/editing instrument 200. For example, a user may open andposition each of the reagent cartridges 210 comprising various desiredinserts and reagents within the chassis of the automated multi-modulecell editing instrument 200 prior to activating cell processing.Further, each of the reagent cartridges 210 may be inserted intoreceptacles in the chassis having different temperature zonesappropriate for the reagents contained therein.

Also illustrated in FIG. 2A is the robotic liquid handling system 258including the gantry 202 and air displacement pipettor 232. In someexamples, the robotic handling system 258 may include an automatedliquid handling system such as those manufactured by Tecan Group Ltd. ofMannedorf, Switzerland, Hamilton Company of Reno, Nev. (see, e.g.,WO2018015544A1), or Beckman Coulter, Inc. of Fort Collins, Colo. (see,e.g., US20160018427A1). Pipette tips 215 may be provided in a pipettetransfer tip supply 214 for use with the air displacement pipettor 232.The robotic liquid handling system allows for the transfer of liquidsbetween modules without human intervention.

Inserts or components of the reagent cartridges 210, in someimplementations, are marked with machine-readable indicia (not shown),such as bar codes, for recognition by the robotic handling system 258.For example, the robotic liquid handling system 258 may scan one or moreinserts within each of the reagent cartridges 210 to confirm contents.In other implementations, machine-readable indicia may be marked uponeach reagent cartridge 210, and a processing system (not shown, but seeelement 237 of FIG. 2B) of the automated multi-module cell editinginstrument 200 may identify a stored materials map based upon themachine-readable indicia. In the embodiment illustrated in FIG. 2A, acell growth module comprises a cell growth vial 218 (for details, seeU.S. Pat. Nos. 10,435,662; 10,433,031; 10,590,375; 10,717,959; and10,883,095). Additionally seen is a tangential flow filtration (TFF)module 222 (for details, see U.S. Ser. Nos. 16/516,701 and 16/798,302).Also illustrated as part of the automated multi-module cell processinginstrument 200 of FIG. 2A is a singulation module 240 (e.g., a solidwall isolation, incubation and normalization device (SWIIN device))shown here and described in detail in U.S. Pat. Nos. 10,533,152;10,633,626; 10,633,627; 10,647,958; 10,723,995; 10,801,008; 10,851,339;10,954,485; 10,532,324; 10,625,212; 10,774,462; and 10,835,869), servedby, e.g., robotic liquid handing system 258 and air displacementpipettor 232. Additionally seen is a selection module 220 which mayemploy magnet separation. Also note the placement of three heatsinks255.

FIG. 2B is a simplified representation of the contents of the exemplarymulti-module cell processing instrument 200 depicted in FIG. 2A.Cartridge-based source materials (such as in reagent cartridges 210),for example, may be positioned in designated areas on a deck of theinstrument 200 for access by an air displacement pipettor 232. The deckof the multi-module cell processing instrument 200 may include aprotection sink (not shown) such that contaminants spilling, dripping,or overflowing from any of the modules of the instrument 200 arecontained within a lip of the protection sink. Also seen are reagentcartridges 210, which are shown disposed with thermal assemblies 211which can create temperature zones appropriate for different reagents indifferent regions. Note that one of the reagent cartridges alsocomprises a flow-through electroporation device 230 (FTEP), served byFTEP interface (e.g., manifold arm) and actuator 231. Also seen is TFFmodule 222 with adjacent thermal assembly 225, where the TFF module isserved by TFF interface (e.g., manifold arm) and actuator 223. Thermalassemblies 225, 235, and 245 encompass thermal electric devices such asPeltier devices, as well as heatsinks, fans and coolers. As in FIG. 2A,gantry 202, tip supply 214, cameras 239 and cooling grate 264 are seen.

The rotating growth vial 218 is within a growth module 234, where thegrowth module is served by two thermal assemblies 235. A selectionmodule is seen at 220. Also seen is the SWIIN module 240, comprising aSWIIN cartridge 244, where the SWIIN module also comprises a thermalassembly 245, illumination 243 (in this embodiment, backlighting),evaporation and condensation control 249, and where the SWIIN module isserved by SWIIN interface (e.g., manifold arm) and actuator 247. Alsoseen in this view is touch screen display 201, display actuator 203,illumination 205 (one on either side of multi-module cell processinginstrument 200), and cameras 239 (one camera on either side ofmulti-module cell processing instrument 200). Finally, element 237comprises electronics, such as a processor, circuit control boards,high-voltage amplifiers, power supplies, and power entry; as well aspneumatics, such as pumps, valves and sensors.

FIG. 2C illustrates a front perspective view of multi-module cellprocessing instrument 200 for use in as a benchtop version of theautomated multi-module cell editing instrument 200. For example, achassis 290 may have a width of about 24-48 inches, a height of about24-48 inches and a depth of about 24-48 inches. Chassis 290 may be andpreferably is designed to hold all modules and disposable supplies usedin automated cell processing and to perform all processes requiredwithout human intervention; that is, chassis 290 is configured toprovide an integrated, stand-alone automated multi-module cellprocessing instrument. As illustrated in FIG. 2C, chassis 290 includestouch screen display 201, cooling grate 264, which allows for air flowvia an internal fan (not shown). The touch screen display providesinformation to a user regarding the processing status of the automatedmulti-module cell editing instrument 200 and accepts inputs from theuser for conducting the cell processing. In this embodiment, the chassis290 is lifted by adjustable feet 270 a, 270 b, 270 c and 270 d (feet 270a-270 c are shown in this FIG. 2C). Adjustable feet 270 a-270 d, forexample, allow for additional air flow beneath the chassis 290.

Inside the chassis 290, in some implementations, will be most or all ofthe components described in relation to FIGS. 2A and 2B, including therobotic liquid handling system disposed along a gantry, reagentcartridges 210 including a flow-through electroporation device, arotating growth vial 218 in a cell growth module 234, a tangential flowfiltration module 222, a SWIIN module 240 as well as interfaces andactuators for the various modules. In addition, chassis 290 housescontrol circuitry, liquid handling tubes, air pump controls, valves,sensors, thermal assemblies (e.g., heating and cooling units) and othercontrol mechanisms. For examples of multi-module cell editinginstruments, see U.S. Pat. Nos. 10,253,316; 10,329,559; 10,323,242;10,421,959; 10,465,185; 10,519,437; 10,584,333; 10,584,334; 10,647,982;10,689,645; 10,738,301; 10,738,663; 10,947,532; 10,894,958; 10,954,512;and 11,034,953, all of which are herein incorporated by reference intheir entirety.

Alternative Embodiment of an Automated Cell Editing Instrument

A bioreactor may be used to grow cells—in particular mammaliancells—off-instrument or to allow for cell growth and recoveryon-instrument; e.g., as one module of a multi-module fully-automatedclosed instrument. Further, the bioreactor supports cellselection/enrichment, via expressed antibiotic markers in the growthprocess or via expressed antibodies coupled to magnetic beads and amagnet associated with the bioreactor. There are many bioreactors knownin the art, including those described in, e.g., WO 2019/046766;10,699,519; 10,633,625; 10,577,576; 10,294,447; 10,240,117; 10,179,898;10,370,629; and 9,175,259; and those available from Lonza Group Ltd.(Basel, Switzerland); Miltenyi Biotec (Bergisch Gladbach, Germany),Terumo BCT (Lakewood, Colo.) and Sartorius GmbH (Gottingen, Germany).

FIG. 3A shows one embodiment of a bioreactor assembly 300 suitable forcell growth, transfection, and editing as one component of an automatedmulti-module cell processing instrument. Unlike most bioreactors thatare used to support fermentation or other processes with an eye toharvesting the products produced by organisms grown in the bioreactor,the present bioreactor (and the processes performed therein) isconfigured to grow cells, monitor cell growth (via, e.g., optical meansor capacitance), passage cells, select cells, transfect cells, andsupport the growth and harvesting of edited cells. Bioreactor assembly300 comprises cell growth vessel 301 comprising a main body 304 with alid assembly 302 comprising ports 308, including a motor integrationport 310 configured to accommodate a motor to drive impeller 306 viaimpeller shaft 352. The tapered shape of main body 304 of the growthvessel 301 along with, in some embodiments, dual impellers allows forworking with a larger dynamic range of volumes, such as, e.g., up to 500ml and as low as 100 ml for rapid sedimentation of the microcarriers.

Bioreactor assembly 300 further comprises bioreactor stand assembly 303comprising a main body 312 and growth vessel holder 314 comprising aheat jacket or other heating means (not shown) into which the main body304 of growth vessel 301 is disposed in operation. The main body 304 ofgrowth vessel 301 is biocompatible and preferably transparent—in someembodiments, in the UV and IR range as well as the visible spectrum—sothat the growing cells can be visualized by, e.g., cameras or sensorsintegrated into lid assembly 302 or through viewing apertures or slots346 in the main body 312 of bioreactor stand assembly 303. Camera mountsare shown at 344.

Bioreactor assembly 300 supports growth of cells from a 500,000 cellinput to a 10 billion cell output, or from a 1 million cell input to a25 billion cell output, or from a 5 million cell input to a 50 billioncell output or combinations of these ranges depending on, e.g., the sizeof main body 304 of growth vessel 301, the medium used to grow thecells, the type and size and number of microcarriers used for growth (ifmicrocarriers are used), and whether the cells are adherent ornon-adherent. The bioreactor that comprises assembly 300 supports growthof both adherent and non-adherent cells, wherein adherent cells aretypically grown of microcarriers as described in detail in U.S. Ser. No.17/237,747, filed 24 Apr. 2021. Alternatively, another option forgrowing mammalian cells in the bioreactor described herein is growingsingle cells in suspension using a specialized medium such as thatdeveloped by ACCELLTA™ (Haifa, Israel). Cells grown in this medium mustbe adapted to this process over many cell passages; however, onceadapted the cells can be grown to a density of >40 million cells/ml andexpanded 50-100× in approximately a week, depending on cell type.

Main body 304 of growth vessel 301 preferably is manufactured byinjection molding, as is, in some embodiments, impeller 306 and theimpeller shaft 352. Impeller 306 also may be fabricated from stainlesssteel, metal, plastics or the polymers listed infra. Injection moldingallows for flexibility in size and configuration and also allows for,e.g., volume markings to be added to the main body 304 of growth vessel301. Additionally, material from which the main body 304 of growthvessel 301 is fabricated should be able to be cooled to about 4° C. orlower and heated to about 55° C. or higher to accommodate cell growth.Further, the material that is used to fabricate the vial preferably isable to withstand temperatures up to 55° C. without deformation.Suitable materials for main body 304 of growth vessel 301 include cyclicolefin copolymer (COC), glass, polyvinyl chloride, polyethylene,polyetheretherketone (PEEK), polypropylene, polycarbonate, poly(methylmethacrylate (PMMA)), polysulfone, poly(dimethylsiloxane), cyclo-olefinpolymer (COP), and co-polymers of these and other polymers. Preferredmaterials include polypropylene, polycarbonate, or polystyrene. Thematerial used for fabrication may depend on the cell type to be grown,transfected and edited, and be conducive to growth of both adherent andnon-adherent cells and workflows involving microcarrier-basedtransfection. The main body 304 of growth vessel 301 may be reusable or,alternatively, may be manufactured and configured for a single use. Inone embodiment, main body 304 of growth vessel 301 may support cellculture volumes of 25 ml to 500 ml, but may be scaled up to support cellculture volumes of up to 3 L.

The bioreactor stand assembly comprises a stand or frame 350 and a mainbody 312 that holds the growth vessel 301 during operation. Thestand/frame 350 and main body 312 are fabricated from stainless steel,other metals, or polymer/plastics. The bioreactor stand assembly mainbody further comprises a heat jacket (not seen in FIG. 3A) to maintainthe growth vessel main body 304—and thus the cell culture—at a desiredtemperature. Additionally, the stand assembly can host a set of sensorsand cameras (camera mounts are shown at 344) to monitor cell culture.

FIG. 3B depicts a top-down view of one embodiment of vessel lid assembly302. Growth vessel lid assembly 302 is configured to be air-tight,providing a sealed, sterile environment for cell growth, transfectionand editing as well as to provide biosafety in a closed system. Vessellid assembly 302 and the main body of growth vessel can be reversiblysealed via fasteners such as screws, or permanently sealed usingbiocompatible glues or ultrasonic welding. Vessel lid assembly 302 insome embodiments is fabricated from stainless steel such as S316Lstainless steel but may also be fabricated from metals, other polymers(such as those listed supra) or plastics. As seen in this FIG. 3B—aswell as in FIG. 3A—vessel lid assembly 302 comprises a number ofdifferent ports to accommodate liquid addition and removal; gas additionand removal; for insertion of sensors to monitor culture parameters(described in more detail infra); to accommodate one or more cameras orother optical sensors; to provide access to the main body 304 of growthvessel 301 by, e.g., a liquid handling device; and to accommodate amotor for motor integration to drive one or more impellers 306.Exemplary ports depicted in FIG. 3B include three liquid-in ports 316(at 4 o'clock, 6 o'clock and 8 o'clock); two self-sealing ports 317, 330(at 3 o'clock and at 7 o'clock) to provide access to the main body 304of growth vessel 301; one liquid-out port 322 (at 11 o'clock); acapacitance sensor 318 (at 9 o'clock); one “gas in” port 324 (at 12o'clock); one “gas out” port 320 (at 10 o'clock); an optical sensor 326(at 1 o'clock); a rupture disc 328 at 2 o'clock; and (a temperatureprobe 332 (at 5 o'clock).

The ports shown in vessel lid assembly 302 in this FIG. 3B are exemplaryonly and it should be apparent to one of ordinary skill in the art giventhe present disclosure that, e.g., a single liquid-in port 316 could beused to accommodate addition of all liquids to the cell culture ratherthan having a liquid-in port for each different liquid added to the cellculture. Further, any liquid-in port may serve as both a liquid-in portand a liquid-out port. Similarly, there may be more than one gas-in port324, such as one for each gas, e.g., O₂, CO₂ that may be added. Inaddition, although a temperature probe 332 is shown, a temperature probealternatively may be located on the outside of vessel holder 314 ofbioreactor stand assembly 303 separate from or integrated into heaterjacket (not seen in this FIG. 3B). A self-sealing port 330, if present,allows access to the main body 304 of growth vessel 301 for, e.g., apipette, syringe, or other liquid delivery system via a gantry (notshown). As shown in FIG. 3A, additionally there may be a motorintegration port 310 to drive the impeller(s), although otherconfigurations of growth vessel 301 may alternatively integrate themotor drive at the bottom of the main body 304 of growth vessel 301.Growth vessel lid assembly 302 may also comprise a camera port forviewing and monitoring the cells.

Additional sensors include those that detect dissolved O₂ concentration,dissolved CO₂ concentration, culture pH, lactate concentration, glucoseconcentration, biomass, and optical density. The sensors may use optical(e.g., fluorescence detection), electrochemical, or capacitance sensingand either be reusable or configured and fabricated for single-use.Sensors appropriate for use in the bioreactor are available from OmegaEngineering (Norwalk Conn.); PreSens Precision Sensing (Regensburg,Germany); C-CIT Sensors AG (Waedenswil, Switzerland), and ABERInstruments Ltd. (Alexandria, Va.). In one embodiment, optical densityis measured using a reflective optical density sensor to facilitatesterilization, improve dynamic range and simplify mechanical assembly.

The rupture disc, if present, provides safety in a pressurizedenvironment, and is programmed to rupture if a threshold pressure isexceeded in growth vessel. If the cell culture in the growth vessel is aculture of adherent cells, microcarriers may be used as described inU.S. Ser. No. 17/237,747, filed 24 Apr. 2021. In such an instance, theliquid-out port may comprise a filter such as a stainless steel orplastic (e.g., polyvinylidene difluoride (PVDF), nylon, polypropylene,polybutylene, acetal, polyethylene, or polyamide) filter or frit toprevent microcarriers from being drawn out of the culture during, e.g.,medium exchange, but to allow dead cells to be withdrawn from thevessel. Additionally, a liquid port may comprise a filter sipper toallow cells that have been dissociated from microcarriers to be drawninto the cell corral while leaving spent microcarriers in main body 304of growth vessel 301. The microcarriers used for initial cell growth canbe nanoporous (where pore sizes are typically <20 nm in size),microporous (with pores between >20 nm to <1 μm in size), or macroporous(with pores between >1 μm in size, e.g. 20 μm) and the microcarriers aretypically 50-200 μm in diameter; thus the pore size of the filter orfrit in the liquid-out port will differ depending on microcarrier size.

The microcarriers used for cell growth depend on cell type and desiredcell numbers, and typically include a coating of a natural or syntheticextracellular matrix or cell adhesion promoters (e.g., antibodies tocell surface proteins or poly-L-lysine) to promote cell growth andadherence. Microcarriers for cell culture are widely commerciallyavailable from, e.g., Millipore Sigma, (St. Louis, Mo., USA);ThermoFisher Scientific (Waltham, Mass., USA); Pall Corp. (PortWashington, N.Y., USA); GE Life Sciences (Marlborough, Mass., USA); andCorning Life Sciences (Tewkesbury, Mass., USA). As for the extracellularmatrix, natural matrices include collagen, fibrin and vitronectin(available, e.g., from ESBio, Alameda, Calif., USA), and syntheticmatrices include MATRIGEL® (Corning Life Sciences, Tewkesbury, Mass.,USA), GELTREX™ (ThermoFisher Scientific, Waltham, Mass., USA), CULTREX®(Trevigen, Gaithersburg, Md., USA), biomemetic hydrogels available fromCellendes (Tubingen, Germany); and tissue-specific extracellularmatrices available from Xylyx (Brooklyn, N.Y., USA); further,denovoMatrix (Dresden, Germany) offers screenMATRIX™, a tool thatfacilitates rapid testing of a large variety of cell microenvironments(e.g., extracellular matrices) for optimizing growth of the cells ofinterest.

FIG. 3C is a side perspective view of the assembled bioreactor 342without sensors mounted in ports 308. Seen are vessel lid assembly 302,bioreactor stand assembly 303, bioreactor stand main body 312 into whichthe main body of growth vessel 301 (not seen in this FIG. 3C) isinserted. Also present are two camera mounts 344, a motor integrationport 310, and stand or frame 350.

FIG. 3D shows the embodiment of a bioreactor/cell corral assembly 360,comprising the bioreactor assembly 300 for cell growth, transfection,and editing described in FIG. 3A and further comprising a cell corral361. Bioreactor assembly 300 comprises a growth vessel 301 (not labeledin this FIG. D) comprising tapered a main body 304 with a lid assembly302 comprising ports 308 (here, 308 a, 308 b, 308 c), including a motorintegration port 310 driving impeller 306 via impeller shaft 352, aswell as two viewing ports 346. Cell corral 361 comprises a main body364, and end caps, where the end cap proximal the bioreactor assembly300 is coupled to a filter sipper 362 comprising a filter portion 363disposed within the main body 304 of the bioreactor assembly 300. Thefilter sipper is disposed within the main body 304 of the bioreactorassembly 300 but does not reach to the bottom surface of the bioreactorassembly 300 to leave a “dead volume” for spent microcarriers to settlewhile cells are removed from the growth vessel 301 into the cell corral361. The cell corral may or may not comprise a temperature or CO₂ probe,and may or not be enclosed within an insulated jacket.

The cell corral 361, like the main body 304 of growth vessel 301 isfabricated from any biocompatible material such as polycarbonate, cyclicolefin copolymer (COC), glass, polyvinyl chloride, polyethylene,polyetheretherketone (PEEK), polypropylene, poly(methyl methacrylate(PMMA)), polysulfone, poly(dimethylsiloxane), cyclo-olefin polymer(COP), and co-polymers of these and other polymers. Likewise, the endcaps of the cell corral are fabricated from a biocompatible materialsuch as polycarbonate, cyclic olefin copolymer (COC), glass, polyvinylchloride, polyethylene, polyetheretherketone (PEEK), polypropylene,poly(methyl methacrylate (PMMA)), polysulfone, poly(dimethylsiloxane),cyclo-olefin polymer (COP), and co-polymers of these and other polymers.The cell corral may be coupled to or integrated with one or moredevices, such as a flow cell where an aliquot of the cell culture can becounted. Additionally, the cell corral may comprise additional liquidports for adding medium, other reagents, and/or fresh microcarriers tothe cells in the cell corral. The volume of the main body 364 of thecell corral 361 may be from 25 to 3000 mL, or from 250 to 1000 mL, orfrom 450 to 500 mL.

In operation, the bioreactor/cell corral assembly 360 comprising thebioreactor assembly 300 and cell corral 361 grows, passages, transfects,and supports editing and further growth of mammalian cells (note, thebioreactor stand assembly is not shown in this FIG. 3D). Cells aretransferred to the growth vessel 301 comprising medium andmicrocarriers. The cells are allowed to adhere to the microcarries.Approximately 2000,000 microcarriers (e.g., laminin-521 coatedpolystyrene with enhanced attachment surface treatment) are used for theinitial culture of approximately 20 million cells to where there areapproximately 50 cells per microcarrier. The cells are grown until thereare approximately 500 cells per microcarrier. For medium exchange, themicrocarriers comprising the cells are allowed to settle and spentmedium is aspirated via a sipper filter, wherein the filter has a meshsmall enough to exclude the microcarriers. The mesh size of the filterwill depend on the size of the microcarriers and cells present buttypically is from 50 to 500 μm, or from 70 to 200 μm, or from 80 to 110μm. For passaging the cells, the microcarriers are allowed to settle andspent medium is removed from the growth vessel 301, and phosphobufferedsaline or another wash agent is added to the growth vessel 301 to washthe cells on the microcarriers. Optionally, the microcarriers areallowed to settle once again, and some of the wash agent is removed. Atthis point, the cells are dissociated from the microcarriers.Dissociation may be accomplished by, e.g., bubbling gas or air throughthe wash agent in the growth vessel 301, by increasing the impellerspeed and/or direction, by enzymatic action (via, e.g., trypsin), or bya combination of these methods. In one embodiment, a chemical agent suchas the RelesR™ reagent (STEMCELL Technologies Canada INC., Vancouver,BC) is added to the microcarriers in the remaining wash agent for aperiod of time required to dissociate most of the cells from themicrocarriers, such as from 1 to 60 minutes, or from 3 to 25 minutes, orfrom 5 to 10 minutes. Once enough time has passed to dissociate thecells, cell growth medium is added to the growth vessel 301 to stop theenzymatic reaction.

Once again, the now-spent microcarriers are allowed to settle to thebottom of the growth vessel 301 and the cells are aspirated through afilter sipper into the cell corral 361. The growth vessel 301 isconfigured to allow for a “dead volume” of 2 mL to 200 mL, or 6 mL to 50mL, or 8 mL to 12 mL below which the filter sipper does not aspiratemedium to ensure the settled spent microcarriers are not transported tothe filter sipper during fluid exchanges. Once the cells are aspiratedfrom the bioreactor vessel leaving the “dead volume” of medium and spentmicrocarriers, the spent microcarriers are aspirated through anon-filter sipper into waste. The spent microcarriers (and thebioreactor vessel) are diluted in phosphobuffered saline or other bufferone or more times, wherein the wash agent and spent microcarrierscontinue to be aspirated via the non-filter sipper leaving a cleanbioreactor vessel. After washing, fresh microcarriers or RBMCs and freshmedium are dispensed into the bioreactor vessel and the cells in thecell corral are dispensed back into the bioreactor vessel for anotherround of passaging or for transfection and editing, respectively.

FIG. 3E depicts a bioreactor and bioreactor/cell corral assembly 360comprising a growth vessel 301, with a main body 364, lid assembly 302comprising a motor integration port 310, a filter sipper 362 comprisinga filter 363 and a non-filter sipper 371, 368. Also seen is a cellcorral 361, fluid line 368 from the cell corral through pinch valve 366,and a line 369 for medium exchange also connected to a pinch valve 366.The non-filter sipper 368 also runs through a pinch valve 366 to waste365. Also seen is a peristaltic pump 367.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example I: Fully-Automated Singleplex RGN-Directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument of thedisclosure. For examples of multi-module cell editing instruments, seeU.S. Pat. No. 10,253,316, issued 9 Apr. 2019; U.S. Pat. No. 10,329,559,issued 25 Jun. 2019; U.S. Pat. No. 10,323,242, issued 18 Jun. 2019; U.S.Pat. No. 10,421,959, issued 24 Sep. 2019; U.S. Pat. No. 10,465,185,issued 5 Nov. 2019; U.S. Pat. No. 10,519,437, issued 31 Dec. 2019; U.S.Pat. No. 10,584,333, issued 10 Mar. 2020; U.S. Pat. No. 10,584,334,issued 10 Mar. 2020; U.S. Pat. No. 10,647,982, issued 12 May 2020; U.S.Pat. No. 10,689,645, issued 23 Jun. 2020; U.S. Pat. No. 10,738,301,issued 11 Aug. 2020; and U.S. Ser. No. 16/920,853, filed 6 Jul. 2020;and Ser. No. 16/988,694, filed 9 Aug. 2020, all of which are hereinincorporated by reference in their entirety.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via Gibson Assembly® into an “editing vector” in an isothermalnucleic acid assembly module included in the automated instrument.lacZ_F172 functionally knocks out the lacZ gene. “lacZ_F172*” indicatesthat the edit happens at the 172nd residue in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The assembled editing vector andrecombineering-ready, electrocompetent E. Coli cells were transferredinto a transformation module for electroporation. The cells and nucleicacids were combined and allowed to mix for 1 minute, and electroporationwas performed for 30 seconds. The parameters for the poring pulse were:voltage, 2400 V; length, 5 ms; interval, 50 ms; number of pulses, 1;polarity, +. The parameters for the transfer pulses were: Voltage, 150V; length, 50 ms; interval, 50 ms; number of pulses, 20; polarity, +/−.Following electroporation, the cells were transferred to a recoverymodule (another growth module), and allowed to recover in SOC mediumcontaining chloramphenicol. Carbenicillin was added to the medium after1 hour, and the cells were allowed to recover for another 2 hours. Afterrecovery, the cells were held at 4° C. until recovered by the user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0E^(0.3)total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example II: Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing system. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via Gibson Assembly® into an“editing vector” in an isothermal nucleic acid assembly module includedin the automated system. Similar to the lacZ_F172 edit, the lacZ_V10edit functionally knocks out the lacZ gene. “lacZ_V10” indicates thatthe edit happens at amino acid position 10 in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The first assembled editing vectorand the recombineering-ready electrocompetent E. coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD600 of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in anisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y145* cassette introduces a stop codon at the154th amino acid reside, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product non-functional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell processingsystem.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

I claim:
 1. A method for multiplex insertion of large DNA payloads into a population of cells and identifying cells with a desired phenotype or genotype comprising the steps of: designing and synthesizing a library of editing cassettes comprising landing pads, wherein the editing cassettes further comprise a gRNA comprising homology to a target sequence in the cells and a repair template comprising 5′ and 3′ homology arms flanking the landing pad; inserting the library of editing cassettes into a vector backbone resulting in a library of editing vectors; transforming the population of cells with the library of editing vectors; allowing editing to take place in the population of cells to produce edited cells; transforming the edited cells with vectors carrying large DNA payloads, wherein the vectors carrying large DNA payloads further comprise a coding sequence for a recombinase or a meganuclease under control of an inducible promoter; inducing expression of the recombinase or meganuclease to insert the large DNA payloads into the landing pads; and screening for cells comprising the desired phenotype or genotype.
 2. The method of claim 1, wherein the vectors carrying large DNA payloads comprise a coding sequence for a recombinase, the landing pads comprise a recognition sequence for the recombinase and the large DNA payloads comprise recognition sequences for the recombinase flanking the large DNA payload.
 3. The method of claim 2, wherein the recombinase is a cyclization recombination enzyme (Cre) and the landing pad and large DNA payload comprise lox recombination sites.
 4. The method of claim 2, wherein the recombinase is flippase and the landing pad and large DNA payload comprise flippase recognition targets (FRTs).
 5. The method of claim 1, wherein the vectors carrying large DNA payloads comprise a coding sequence for a meganuclease, the landing pads comprise a recognition sequence for the meganuclease, and the large DNA payloads further comprise homologous recombination sequences flanking the DNA playloads.
 6. The method of claim 5, wherein the meganuclease belongs to the LAGLIDADG family of nucleases.
 7. The method of claim 6, wherein the meganuclease is I-SceI.
 8. The method of claim 6, wherein the meganuclease is I-CreI.
 9. The method of claim 6, wherein the meganuclease is I-DmoI.
 10. The method of claim 1, wherein the editing cassettes further comprise a barcode.
 11. The method of claim 1, wherein the editing cassettes further comprise an amplification priming site at the 3′ end of the editing cassette.
 12. The method of claim 1, wherein the vectors carrying the large DNA payloads further comprise a selectable marker and the method further comprises a selection step between the transforming and allowing steps.
 13. The method of claim 12, wherein the selectable marker in the vectors carrying the large DNA payloads is different from a selectable marker in the editing cassettes.
 14. The method of claim 1, wherein the vectors carrying the large DNA payloads comprise the coding sequence of the recombinase or meganuclease under the control of an inducible primer.
 15. The method of claim 14, wherein the inducible promoter is a pL promoter.
 16. The method of claim 14, wherein the inducible promoter is a pBAD promoter.
 17. The method of claim 1, wherein the vectors carrying large DNA payloads further comprise an origin of replication and a selectable marker.
 18. The method of claim 1, wherein the large DNA payloads are from 100 bp to 100 Kb in length.
 19. The method of claim 18, wherein the large DNA payloads are from 500 bp to 50 Kb in length.
 20. The method of claim 1, wherein the screening step comprises polymerase chain reaction (PCR) analysis with appropriate primer sets; a metabolic test; measurement of transcript level; a phenotypic assay; detection of a protein product using an antibody specific to the protein product; or DNA sequencing of the integrated large DNA payload.
 21. A method for multiplex insertion of large DNA payloads into a population of cells and identifying cells with a desired phenotype or genotype comprising the steps of: designing and synthesizing a library of editing cassettes comprising landing pads wherein the landing pads comprise a recognition sequence for a recombinase and wherein the editing cassettes further comprise a gRNA comprising homology to a target sequence in the cells and a repair template comprising 5′ and 3′ homology arms flanking the landing pad; inserting the library of editing cassettes into a vector backbone resulting in a library of editing vectors; transforming the population of cells with the library of editing vectors; allowing editing to take place in the population of cells to produce edited cells; transforming the edited cells with vectors carrying large DNA payloads, wherein the vectors carrying large DNA payloads further comprise a coding sequence for the recombinase under control of an inducible promoter; inducing expression of the recombinase to insert the large DNA payloads into the landing pads; and screening for cells comprising the desired phenotype or genotype.
 22. The method of claim 21, wherein the recombinase is a cyclization recombination enzyme (Cre) and the landing pad and large DNA payload comprise lox recombination sites.
 23. The method of claim 21, wherein the recombinase is flippase and the landing pad and large DNA payload comprise flippase recognition targets (FRTs).
 24. The method of claim 21, wherein the editing cassettes further comprise an amplification priming site at the 3′ end of the editing cassette and wherein the vectors carrying the large DNA payloads further comprise a selectable marker and the method further comprises a selection step between the transforming and allowing steps.
 25. A method for multiplex insertion of large DNA payloads into a population of cells and identifying cells with a desired phenotype or genotype comprising the steps of: designing and synthesizing a library of editing cassettes comprising landing pads wherein the landing pads comprise a recognition sequence for a meganuclease and wherein the editing cassettes further comprise a gRNA comprising homology to a target sequence in the cells and a repair template comprising 5′ and 3′ homology arms flanking the landing pad; inserting the library of editing cassettes into a vector backbone resulting in a library of editing vectors; transforming the population of cells with the library of editing vectors; allowing editing to take place in the population of cells to produce edited cells; transforming the edited cells with vectors carrying large DNA payloads, wherein the vectors carrying large DNA payloads further comprise a coding sequence for the meganuclease under control of an inducible promoter; inducing expression of the meganuclease to insert the large DNA payloads into the landing pads; and screening for cells comprising the desired phenotype or genotype.
 26. The method of claim 24, wherein the meganuclease belongs to the LAGLIDADG family of nucleases.
 27. The method of claim 25, wherein the meganuclease is I-SceI.
 28. The method of claim 25, wherein the meganuclease is I-CreI.
 29. The method of claim 25, wherein the meganuclease is I-DmoI.
 30. The method of claim 24, wherein the editing cassettes further comprise an amplification priming site at the 3′ end of the editing cassette and wherein the vectors carrying the large DNA payloads further comprise a selectable marker and the method further comprises a selection step between the transforming and allowing steps. 